It is known that Pseudomonas fluorescens strains are able to biosynthesize, in addition to pseudomonic acid A, other related antibiotics designated by the letters B-D in small quantities [E. B. Chain, G. Mellows, J. Chem. Soc. Perkin Trans I. 318 (1977); J. P. Clayton et al., Tetrahedron Lett., 21, 881 (1980); P. J. O. Hanlon, N. H. Rogers, J. Chem. Soc. Perkin Trans I. 2665 (1983)]. Among the pseudomonic acid antibiotics, from a therapeutic point of view the most valuable is pseudomonic acid A, which has a growth inhibiting effect mainly against Gram positive bacteria.
One method for the isolation of pseudomonic acid A from the antibiotic complex-containing culture broth is the liquid-liquid extraction. According to German Patent No. 2227739 and U.S. Pat. No. 4,289,703, soluble barium salts are added to the fermentation broth, then the microorganism cells with the insoluble inactive agents are separated by centrifugation and finally the antibiotics are extracted by methyl isobutyl ketone (MIBK). The antibiotics are then removed from the methyl isobutyl ketone extract by alkaline water and the resulting alkaline aqueous extract is cleaned by reextraction with methyl isobutyl ketone. The crude product obtained is chromatographed, and an ester derivative is prepared from the pseudomonic acid antibiotic complex and purified with preparative thin layer chromatography. The acid form of the pure antibiotic is obtained by hydrolysis.
Belgian Patent No. 870,855 relates to a process in which the culture broth is extracted with methyl isobutyl ketone and from the extract the active substance is extracted by sodium hydrogen carbonate solution. Materials insoluble in alkaline water are separated by filtration, then the pH of the filtrate is acidified and extracted by methyl isobutyl ketone. Finally the pseudomonic acid A is obtained by the concentration of the extract and crystallization from a methyl isobutyl ketone-n-heptane mixture.
U.S. Pat. No. 4,222,942 relates to a process for the isolation of mupirocin by extracting a solution of crude mupirocin preparation in a water immiscible organic solvent with an aqueous media, lowering the pH of said aqueous solution, and extracting the solution with a polar water immiscible organic solvent and thereafter adding a diluent which is sufficiently non-polar to reduce the polarity of, but is miscible with, said polar organic solvent so as to effect crystallization of mupirocin. The steps of this reaction scheme are manifold and, as a consequence, the scheme requires a large quantity of solvent.
U.S. Pat. No. 6,254,921 discloses a process for the isolation of mupirocin comprising extracting an acidified culture broth using a chlorinated aliphatic hydrocarbon or isobutyl acetate, such that a mupirocin-containing extract is obtained; and purifying the mupirocin from said extract by distributing the extract between an aqueous phase and an organic phase comprising at least one organic solvent and evaporating the organic solvent.
None of the above methods are satisfactory from a commercial and ecological view. Accordingly, there remains a need for a novel method for purification of mupirocin, which is free from at least some of the disadvantages of the known processes and the application of which, in production scale, may result in a high yield of the recovery of the above-mentioned antibiotic.
The present invention provides a method for purification of mupirocin, which meets at least some of the above defined objectives in that it is economically sound and, preferably, less ecologically deleterious, and results in a high yield of mupirocin having a high purity. The method comprises precipitation of mupirocin from an aqueous solution of mupirocin, and dissolution of the precipitate in an organic solvent.
The present invention relates to a method for purifying mupirocin, comprising the steps of:                a) precipitation of mupirocin from an aqueous solution of mupirocin;        b) recovery of the precipitate;        c) dissolution of the precipitate in an organic solvent; and optionally        ci) recovery of mupirocin from the solvent.        
The term “mupirocin” is meant to comprise mupirocin as defined in any of the above-mentioned references, as well as natural occurring and synthetic derivatives thereof. The term comprises the acidic form and salts thereof, such as the calcium salt, as well as solvates and polymorphic forms of these.
In an embodiment of the invention, the solution of mupirocin is fermentation broth, such as clarified broth, such as clarified by centrifugation or filtration. The source of the fermentation broth is not crucial, and the broth can be obtained as disclosed in the prior art, for example as disclosed in the above-mentioned references, in U.S. Pat. Nos. 3,977,943, 4,071,536 and 4,289,703, WO 00/46389 or WO 03/000910. Optionally, the broth can be concentrated before precipitation of mupirocin. Solutions other than fermentation broths can be used, e.g. a solution resulting from chemical synthesis of mupirocin or a derivative thereof.
Mupirocin may be precipitated from the aqueous solution by adding an acid, such as an acid selected from the group consisting of acetic acid; citric acid; sulfuric acid; and hydrochloric acid. When using an acid, the pH during precipitation conveniently is in the range of 2.0 to 6.0, preferably in the range of 3.5 to 5.0, and most preferred in the range of 4.2 to 4.6. In one embodiment the solution is left stirring until mupirocin has precipitated, such as for a period of 10-40 minutes.
The mupirocin containing precipitate may be recovered by centrifugation or filtration, centrifugation being preferred. The centrifugation should be carried out under such conditions that a gel is formed, e.g. by centrifuging for 5-30 minutes at 5000-15000 g, or in an continuous centrifuge at 5000-15000 g.
In an embodiment of the invention, the recovered precipitate is dissolved in a polar, substantially water immiscible organic solvent. As solvent, a ketone, e.g. having the formula alkyl-C(═O)-alkyl, such as methyl isobutyl ketone; an ester, e.g. having the formula alkyl-C(═O)—O-alkyl, such as ethyl acetate; an alkanol, e.g. having the formula alkyl-OH, such as n-butanol; or a mixture comprising any of these can be used. Herein, the term “alkyl” when referring to alcohols, refers to aliphatic hydrocarbon groups of greater than four carbons and includes a straight or branched chain aliphatic hydrocarbon group that is saturated or unsaturated and, in other solvents, the term “alkyl” refers to a straight or branched chain aliphatic hydrocarbon group which is saturated or unsaturated and has 1, 2, 3, 4, 5, 6 or more carbon atoms.
Presently it is preferred that the solvent is pure methyl isobutyl ketone (MIBK). That is the solvent should comprise more that 90% (such as more than 92%, more than 94%, more than 96%, or even more than 98% or 99%) MIBK. If needed, the mupirocin can be recovered from the solvent, such as by crystallization, optionally after water removal from the solvent.
In another embodiment, the method of the invention comprises one or more further steps, in order to obtain a more pure antibiotic. Such steps might be applied before step a) and/or after step d), and/or between steps a) and b) and/or between steps b) and c) and/or after step c), and comprises steps selected from the group consisting of: a decolourisation step; a step for removal of hydrophilic components; a concentration step; an evaporation step; a clarification step; a water removal step; a drying step; a filtration step; an extraction step; and a crystallization step. It is presently preferred that the further step(s) are applied after the step d) above.
The method of the invention is preferably carried out at a temperature between the freezing point of the solutions and room temperature, such as a temperature in the range 0-25° C., more preferred in the range 5-20° C. Optionally drying and evaporation steps can be carried out at an elevated temperature, such as in the range 20-50° C.
The invention also relates to mupirocin obtained by a method of the invention. The mupirocin of the invention is more pure than mupirocin obtained by the methods of the prior art. By the method of the invention, it is possible to obtain mupirocin having a purity of at least 93%, for example at least 95%, preferably least 96%, more preferably least 97% and most preferably even at least 98%. Typically, the mupirocin of the invention has a specific activity of at least 950 μg/mg, and preferably at least 960 μg/mg.
Following the teaching of the prior art (e.g. U.S. Pat. Nos. 5,569,672 and 5,594,026, the entire disclosure of which is incorporated by reference herein) it is also possible to generate calcium salts and different polymorphic forms of pseudomonic acid.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All values mentioned herein should be understood as being preceded by “about”, i.e. “7” should be understood as “about 7”. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. The content of the prior art references are hereby incorporated herein in their entirety by reference.